Plant transformation is now a core research tool in plant biology and a practical tool for cultivar improvement; transformation of several plant species via Agrobacterium mediated transformation has become a routine technology. Sunflower is a major crop used worldwide for the production of edible oils; attempts to transform the species are being made for a number of decades—however, the sunflower is a recalcitrant species resistant to the methods of transformation used so far. Attempts to transform the species using Agrobacterium or biolistic methods, using different selectable markers have resulted in very low transformation efficiencies ranging from 0.6-6%.
When a population of plant cells is transformed, selection of the transformed cells is typically done using a selection marker. The major technical challenge in plant transformation is the development of selection markers for screening and selection of successfully transformed cells. This process of selection is extremely important, with the cost of screening for transformants sometimes exceeding the costs of transformation itself. The choice of the selectable marker gene, the selective agent, its concentration and the timing of the application is very important for a strict selection of transformed cells. On the other hand, regeneration should not be impeded. Hence though a strict selection regime is desirable, this should not impede the development potential of the transformants. The choice of conditions that rightly balances the two needs constitutes an art in itself.
Selectable markers identified today can be differentiated into two types that enable transgenic plants or cells to be identified after transformation. They can be divided into positive and negative markers conferring a selective advantage or disadvantage respectively. Negative selectable markers are those, which allow the selection of transformed cells, or tissue explants by their ability to grow in the presence of an antibiotic or herbicide. In addition to selecting of transformants, such markers can be used to follow the inheritance of a foreign gene in segregating population of plants.
These negative selection methods have considerable disadvantages. The most significant of them all is that the non-transformed cells are killed in the presence of the phyto-toxic product and in cases where a coherent tissue is used there is a risk that the transformed cells also die, due to the fact that the death of the non-transformed cells may cut off the supply of nutrients to the transformed cells or because of the damaged or dying non-transformed cells may excrete toxic compounds. Moreover the presence of an antibiotic resistance gene in ingested plants is a matter of concern. In addition, selection of cells or tissues using negative selection requires precise timing of expression of the introduced genes in relation to the selection process. If the transgenic cells are treated with a toxic compound before the detoxifying gene is expressed or before enough of the gene product is produced to ameliorate the action of the toxic compound, both the transgenic and the non-transgenic cells are killed. If selection is delayed, the selection of transgenic cells or tissues may be hindered by, for example, shoot or callus formation from non-transgenic cells or tissues, which forms a barrier to the penetration of the compound used to select the transformed cells.
The above-mentioned disadvantages are overcome to a substantial extent, by the method of positive selection whose operating principle is converse to negative selection. Rather than conferring resistance to a negative or toxic substance, positive selection involves conferring onto the transformed cell a metabolic, or other competitive advantage over non-transformed cells. These identify and select the genetically transformed cells without damaging or killing the non-transformed cells in the population.
Sunflower is an important oil-seed crop, which is reported to be recalcitrant to transformation and regeneration (Schrammeijer et al., Plant Cell Reports, 1990 9:55-60). Schrammeijer et al., have evaluated some of the potential problems with the regeneration from various explants of Sunflower, the transformants being selected by their ability to grow on negative selection based agents. Kanamycin and hygromycin are the most widely used selection agents for sunflower transformation although these are known to be detrimental to their organogenic potential (Everett et al., 1987; Muller et al., 2001).
Disclosed in this invention is a novel method of selecting transformants belonging to the species Helianthus annuus, specifically exemplifying a positive selection method that involves conferring to the transformed tissue explants an ability to metabolize certain compounds preferably Xylose; transformed explants are selected by simply culturing them on a medium containing the referred selection agent.
The method of selection disclosed herein relates to a method of screening and selecting suitable transformed explants for regeneration. A stable, precise, higher transformation and regeneration efficiency has been achieved through our method. The advantages of the technology described are lucid enough to distinguish it from the existing technologies.